Diarylalkanes as potent inhibitors of binuclear enzymes

ABSTRACT

The present invention implements a strategy that combines an enzyme inhibition assay with a chemical dereplication process to identify active plant extracts and the particular compounds—diarylalkanes and/or diarylalkanols within those extracts that specifically inhibit binuclear enzyme function. Included in the present invention are compositions of matter comprised of one or more of diarylalkanes and/or diarylalkanols, which inhibit the activity of binuclear enzymes, particularly tyrosinase and which prevent melanin overproduction. The present invention also provides a method for inhibiting the activity of a binuclear enzyme, particularly tyrosinase and a method for preventing and treating diseases and conditions related to binuclear enzyme function. The present invention further includes a method for preventing and treating melanin overproduction and diseases and conditions of the skin related thereto. The method for preventing and treating diseases and conditions related to binuclear enzyme function and melanin overproduction is comprised of administering to a host in need thereof an effective amount of a composition comprising one or more diarylalkanes and/or diarylalkanols synthesized and/or isolated from one or more plants together with a pharmaceutically acceptable carrier.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/209,814, filed Mar. 13, 2014, now allowed, which is a continuation ofU.S. application Ser. No. 13/707,270, filed Dec. 6, 2012, now issued asU.S. Pat. No. 8,729,136, which is a divisional of U.S. application Ser.No. 12/814,190, filed Jun. 11, 2010, now issued as U.S. Pat. No.8,592,488, which is a divisional of U.S. application Ser. No.12/027,090, filed Feb. 6, 2008, now issued as U.S. Pat. No. 7,767,661,which is a continuation of U.S. application Ser. No. 11/139,200, filedMay 27, 2005, now abandoned, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/575,599, filed May 28,2004. Each of these applications is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

This invention relates generally to the prevention and treatment ofdiseases and conditions mediated by binuclear enzymes. Specifically, thepresent invention includes a method for inhibiting the activity of anenzyme having a binuclear active site. Included in the present inventionare novel compositions comprised of one or more diarylalkane(s). Thediarylalkanes of the present invention can be isolated from one or moreplant sources or can be obtained by organic synthesis. Further includedin the present invention are methods for isolating these compounds froma natural source and methods for synthesizing these compounds. In oneembodiment, the diarylalkanes are obtained by synthetic modification ofa naturally occurring compound isolated from a plant source.

BACKGROUND OF THE INVENTION

There is a great demand for products able to inhibit or preventexcessive pigmentation of the skin. Melanin, the skin's natural pigment,is a nitrogenous polymer synthesized in melanosomes, which aremembrane-bound organelle present within melanocytes. Melanin is producedin varying concentrations, depending on skin type (genetic disposition)and environmental conditions. Melanocytes are cells that occur in thebasal membrane of the epidermis, and account for between 5% and 10% ofthe cellular content (approximately 1200-1500 melanocytes per cm²). Whenstimulated, by factors such as ultraviolet (UV) light melanocytes dividemore rapidly, thereby producing greater quantities of melanin. Themelanin is then transported in mature melanosomes to keratinocytes,within the epidermis where it becomes visible as a brown skin color.

The number of melanocytes in human skin is more or less the same,irrespective of skin color. The color of the skin is largely dependenton the quantity and type of melanin produced (black eumelanin or yellowto reddish-brown pheomelanin). Asians and light-skinned people havelower levels of eumelanin than dark-skinned people, and correspondinglyless protection against the effects of radiation. People with red hairare characterized by pigmentation with pheomelanin, and have little orno photo-protection. Additionally, the distribution of melanin in theskin also varies. In people with light skin, the greater part of thepigment lies in the basal layer, whereas in those with dark skin, themelanin is spread throughout, reaching into the horny layer.

The over production of melanin can cause different types of abnormalskin color, hair color and other diseases and conditions of the skin.There are primarily two conditions related to skin pigmentationdisorders. A darkening of the skin that includes abnormal elevatedmelanin caused by UV exposure and aging; and abnormal distribution ofskin pigments resulting in age spots, liver spots, and drug andwound/disease induced hyperpigmentation (Seiberg et al. (2000) J.Invest. Dermatol. 115:162; Paine et al. (2001) J. Invest. Dermatol.116:587).

Modulators of melanogenesis (the production of melanin) may be designedor chosen to function in a variety of ways as illustrated in FIG. 1 .With reference to FIG. 1 , they may act directly on modulatingmelanosome structure and function prior to melanin synthesis, they mayact by inhibiting the production or function of enzymes, such astyrosinase, which are involved in the synthesis of melanin, they maychanging the ratio of eumelanin/pheomelanin, or they may function bydamping mechanisms responsible for transfer of melanosomes frommelanocyte to keratinocytes. (Briganti et al. (2003) Pigment CellResearch 16:101-110).

Tyrosinase is a key enzyme in the production of melanin. It catalyzesthree reactions: the hydroxylation of tyrosine to3,4-dihydroxyphenylalanine (DOPA), oxidation of DOPA to DOPA quinone andthe oxidation of DHI (5,6-dihydroxyindole) to indole quinone. (Hearinget al. (1991) FASEB 53:515). It has been determined that tyrosinaseneeds both the substrate and divalent metal ions for its catalyticactivity. The processes currently used for inhibiting the synthesis ofmelanin with a view to lightening skin are primarily based on substanceswhich inhibit tyrosinase activity, either directly by interacting withtyrosinase itself, or indirectly e.g., by complexing the necessary metalions.

Tyrosinase belongs to the family of type 3 copper proteins, whichcontain two copper ions at their active site. Studies of the structureof the active site of tyrosinase have revealed that the two copper ionsare closely spaced and each ion is coordinated to three histidinesthrough the N-ε nitrogen atom of its side chain, as illustrated in FIG.2 . (Pfiflher and Lerch (1981) Biochem. 20: 6029; Cuff et al. (1998) J.Mol. Biol. 278:855). The binuclear copper ions can exist in three mainredox forms: the Cu^(I)—Cu^(I) reduced form, the Cu^(II)—O₂—Cu^(II) formwhich reversibly binds to O₂ as the peroxide, and the resting form ofthe enzyme, where the Cu²⁺ ions are normally bridged by a small ligand.It has been determined that the Cu^(II)—O₂—Cu^(II) redox state is key tothe enzymatic activity of tyrosinase. In this state, tyrosinasecatalyzes the introduction of a second hydroxyl group to the orthoposition of a mono-phenol (such as tyrosine) a reaction which is key tothe biosynthesis of melanin.

Any compound, which interferes with the access, ligand formation, or theoxidation of monophenols at the active site of tyrosinase, will be anefficient inhibitor of tyrosinase, potentially resulting in a decreasein the production of melanin and lighter skin color. Generally speaking,the copper ions at the active site of tyrosinase can be easily chelatedwith lone pair electrons on oxygen, nitrogen, sulfur and halogens.(Weder et al. (1999) Inorg. Chem. 38:1736). FIG. 3 illustrates thestructures and mechanisms of action of several known tyrosinaseinhibitors. (Briganti et al. (2003) Pigment Cell Research 16:101-110;Seo et al. (2003) J. Agric. Food Chem. 51:2837).

With reference to FIG. 3 , it can be seen that compounds with structuressimilar to 3,4-dihydroxyphenylalanine (DOPA), such as hydroquinone, bothinhibit tyrosinase and are also melanocytolytic agents. (U.S. Pat. No.5,523,077). For example, arbutin, isolated from the leaves of the commonbearberry, Uvae ursi, is a naturally occurring beta-glucopyranoside ofhydroquinone, which inhibits tyrosinase and effects melanin synthesis inhuman melanocytes. (Chakraborty et al. (1998) Pigment Cell Res. 11:206;U.S. Pat. No. 5,980,904). The mechanism of action for arbutin involvescompetition with L-tyrosine or L-dopa for binding at the active site oftyrosinase. It does not suppress the expression or the synthesis of theprotein. (Maeda and Fukuda (1996) J. Pharmacol. Exp. 276:765). Syntheticarbutin type compounds also strongly inhibit human tyrosinase. (Sugimotoet al. (2003) Chem. Pharm. Bull. 51:798). Kinobeon A, a novel diquinoneisolated from cultured cells of safflower (Carthamus tinctorius L.), hastyrosinase inhibitory activity greater than that of kojic acid.(Kanehira et al. (2003) Planta Med. 69:457). If applied over longperiods of time or in high concentrations hydroquinones can have seriousside effects. Additionally, hydroquinones may lead to permanentde-pigmentation, and thus to increased photosensitivity of the skin whenexposed to UV light.

Better-tolerated skin lightening substances currently being used are ofnatural origin. For example, kojic acid is a natural hydroxyl-γ-pyronederived from carbohydrate solutions containing certain bacteria. Withreference to FIG. 3 , it can be seen that kojic acid is an oxidizedortho-dihydroxyphenol. Kojic acid is known to form strong chelates withmetal ions especially Cu^(II). (Gerard and Hugel (1975) Bull. Soc. Chim.Fr. 42:2404). It is a potent competitive, but slow binding inhibitor oftyrosinase. (Cabanes et al. (1994) J. Pharm. Pharmacol. 46:982). Recentstudies have shown that kojic acid acts as a bridging ligand, bindingstrongly to both the dicopper (II) complex and to the dicopper-dioxygenadduct, thereby preventing the binding of the catechol substrate to theenzyme. (Battaini et al. (2000) JBIC 5:262). Kojic acid and its estershave been patented for use as skin whiteners. (see U.S. Pat. Nos.4,369,174; 4,771,060; 5,824,327; 5,427,775; 4,990,330).

Flavonoids are another class of natural products that have been reportedas inhibitors of tyrosinase. (Shimizu et al. (2000) Planta Med. 66:11;Xie et al. (2003) Biochem. 68:487). Active tyrosinase inhibitors includeflavones (Likhitwitayawuid et al. (2000) Planta Med. 66:275), flavonols(Kubo and Kinst-Hori (1999) J. Agric. Food Chem. 47:4121), prenylnatedflavonoids (Kuniyoshi et al. (2002) Planta Med. 68:79; Son et al. (2003)Planta Med. 69:559; Kim et al. (2003) Biol. Pharm. Bull. 26:1348),flavans (No et al. (1999) Life Sci. 65:PL241; Kim et al. (2004)Biomacromolecules 5:474), and dihydro-chalcones (Shoji et al. (1997)Biosci. Biotechnol. Biochem. 61:1963).

Other types of tyrosinase inhibitors include: phenol derivatives (Sakumaet al. (1999) Arch. Pharm. Res. 22:335; Kerry and Rice-Evans (1999) J.Neurochem. 73:247; Battaini et al. (2002) J. Biol. Chem. 277:44606),benzaldehydes (Kubo and Kinst-Hori (1999) Plant Medica 65:19; Chen etal. (2003) J. Enzyme Inhib. Med. Chem. 18:491; Nihei et al. (2004)Bioorg. Med. Chem. 14:681), benzoic acid derivatives (Curto et al.(1999) Biochem Pharmacol. 57:663; Chen et al. (2003) J. Protein Chem.22:607; Miyazawa et al. (2003) J. Agric. Food Chem. 51:9653; Kubo et al.(2003) Z. Naturforsch [C] 58:713), cupferron (Xie et al. (2003) Int. J.Biochem. Cell Biol. 35:1658), benzodipyran from Glycyrrhiza uralensisroot (Yokota et al. (1998) Pigment Cell Res. 11:335), thiohydroxylcompounds (Park et al. (2003) J. Protein Chem. 22:613), terpenoids (Ohet al. (2002) Planta Med. 68:832), and oxazolodinethione (Seo et al.(1999) Planta Med. 65:683). The most potent known natural tyrosinaseinhibitors are stilbenes (IC₅₀=0.3-5 μM) (Shin et al. (1998) BiochemBiophys. Res. Commun. 243:801; Ohguchi et al. (2003) Biosci. Biotechnol.Biochem. 67:1587), stilbene glycosides (Lida et al. (1995) Planta Med.61:425) and 4-substituted resorcinols (Shimizu et al. (2000) Planta Med.66:11).

A structure/activity study of 4-substituted resorcinols reveals thathydrophobic and less bulky substituents, such as —CH₂C₆H₅, and alkylgroups i.e. —CH₂CH₂CH₃ have the greatest potency with IC₅₀'s of lessthan 10 μM (Shimizu et al. (2000) Planta Med. 66:11). The mechanism ofaction for 4-substituted resorcinols has been characterized asslow-binding competitive inhibition of the oxidation ofDL-β-(3,4-dihydroxyphenyl)alanine (DL-dopa) (Jimenez and Garcia-Carmona(1997) J. Agric. Food Chem. 45:2061) without any further understandingof the metal chelating effects on binuclear copper ions.

Aloe, a member of the Lily family, is an intricate plant that containsmany biologically active substances. (Cohen et al. (1992) in WoundHealing/Biochemical and Clinical Aspects, 1st ed. W B Saunders,Philadelphia). Over 360 species of Aloe are known, most of which areindigenous to Africa. Historically, Aloe products have been used indermatological applications for the treatment of burns, sores and otherwounds. These uses have stimulated a great deal of research inidentifying compounds from Aloe plants that have clinical activity.(See, e.g., Grindlay and Reynolds (1986) J. of Ethnopharmacology16:117-151; Hart et al. (1988) J. of Ethnopharmacology 23:61-71).

Yagi et al. disclose a group of compounds isolated from Aloe,particularly aloesin and one of its derivatives, 2″-O-feruloylaloesin,which are effective inhibitors of tyrosinase. (Yagi et al. (1987) PlantMedica 515-517; Yagi et al. (1977) Z. Naturforsch 32c:731-734). Aloesin,a C-glucosylated 5-methylchromone inhibited human tyrosinase hydroxylaseactivity in a dose dependent manner with an IC₅₀ of 0.92 mM and alsoinhibited DOPA oxidase activity in a dose dependent manner withIC₅₀=0.70 mM compared to kojic acid, which has an IC₅₀=0.41 mM, andarbutin which has an IC₅₀=3.02 mM. Inhibition of tyrosinase enzymaticactivity and consequent melanin formation by aloesin was confirmed in acell-based assay using B16 F1 murine melanoma cells. Melaninbiosynthesis was inhibited by aloesin (IC₅₀=0.167 mM) in a dosedependent manner. (Jones et al. (2002) Pigment. Cell Res. 15:335). Themechanism of action of tyrosinase inhibition for aloe chromones isspeculated as being related to the reduction of copper ions. Bothnatural (U.S. Pat. No. 6,451,357), semi-synthetic (U.S. Pat. Nos.5,801,256; 6,083,976) and formulated aloe chromones (U.S. Pat. No.6,123,959) have been patented for their skin whitening ability.

Ascorbic acid (vitamin C from synthetic and natural sources such ascitrus fruits) and its derivatives have also been utilized for skinwhitening. In most cases, vitamin C is formulated with kojic acid orother tyrosinase inhibitors (U.S. Pat. Nos. 4,919,921; 6,458,379 and5,916,915). Other reported skin whitening compounds include extractsfrom olive plants (U.S. Pat. No. 6,682,763), unsaturated long chainfatty acids (U.S. Pat. No. 6,669,932), curcumins (U.S. Pat. No.6,641,845), enzyme extracts (U.S. Pat. No. 6,514,506), coumestrol (U.S.Pat. No. 6,503,941), hydroxyl carboxylic acids (U.S. Pat. Nos.6,417,226; 6,365,137; 5,609,875; 5,262,153), beta-glucans (U.S. Pat. No.6,251,877), aloe chromones (U.S. Pat. No. 6,083,976), phenylalaninecompounds (U.S. Pat. No. 5,767,158), rutin (U.S. Pat. No. 5,145,782),escinol (U.S. Pat. No. 5,728,683), salicylic acids (U.S. Pat. No.5,700,784), angiogenin (U.S. Pat. No. 5,698,185), mercaptodextran (U.S.Pat. No. 6,077,503), ellagic acid (U.S. Pat. No. 6,066,312), phosphinicacids (U.S. Pat. No. 6,280,715), boron containing compounds (U.S. Pat.No. 5,993,835), plant extracts (from Pueraria, U.S. Pat. No. 6,352,685;Moms, U.S. Pat. Nos. 6,197,304; 6,066,312; and 5,872,254; acerola cherryfermentate, U.S. Pat. No. 5,747,006; furanones, U.S. Pat. No. 5,602,256;and others, U.S. Pat. No. 5,773,014).

Diarylalkanes are a rare class of natural product. To date, there aremore than 179,000 natural compounds listed in the Dictionary of NaturalProducts on CD-ROM (Chapman & Hall/CRC, Version 12:2 Jan. 2004), ofwhich only 82 are diarylpropanes (n=3). Broussonetia papyrifera is adeciduous tree in Moracea family and more than twenty diarylpropaneshave been isolated from this genera alone (Keto et al. (1986) Chem.Pharm. Bull. 34:2448; Ikuta et al. (1986) Chem. Pharm. Bull. 34:1968;Takasugi et al. (1984) Chem. Lett. 689; Gonzalez et al. (1993)Phytochem. 32:433). Bioassay directed fractionation of an extract ofBroussonetia papyrifera yielded four diarylpropanes which did not havearomatase inhibitory activity. (Lee et al. (2001) J. Nat. Prod.64:1286). However, two prenylated diarylpropanes isolated from the sameplant exhibited cytotoxicity against several cancer cell lines (Ko etal. (1999) J. Nat. Prod. 62:164) and broussonin A exhibited anti-fungalactivity (lida et al. (1999) Yakugaku Zasshi. 119:964).

A number of diarylalkanes have also been isolated from the IIyantheraspecies (Myristicaceae). (Alyea et al. (1975) Phytochem. 14:2832; deAlmeida et al. (1979) Phytochem. 18:1015; Braz et al. (1980) Phytochem.19:1195; Diaz et al. (1986) Phytochem. 25:2395). Four dihydrochalconesisolated from Iryanthera lancifolia showed antioxidant activity (Silvaet al. (1999) J. Nat. Prod. 62:1475). A number diarylpropanes have alsobeen were isolated from the Virola species of Myristicaceae. (Braz etal. (1976) Phytochem. 15:567; Hagos et al. (1987) Plant Med. 53:57;Gonzalez et al. (1993) Phytochem. 32:433; Kijjoa et al. (1981)Phytochem. 20:1385; Talukdar et al. (2000) Phytochem. 53:155).

Other diarylpropanes isolated from natural sources include those fromPterocarpus marsupium (Fabaceae) (Rao et al. (1984) Phytochem. 23:897;Maurya et al. (1985) J. Nat. Prod. 48:313), Lindera umbellate(Lauraceae) (Morimoto et al. (1985) Chem. Pharm. Bull. 33:2281),Helichrysum mundii (Compositae) (Bohlmann et al. (1978) Phytochem.17:1935), Viscum angulatum (Loranthaceae) (Lin et al. (2002) J. Nat.Prod. 65:638), those from Acacia tortilis (Leguminosae), which have asmooth muscle relaxing effect (Hagos et al. (1987) Planta Med. 53:27),Xanthocercis zambesiaca (Leguminosae) (Bezuidenhout et al. (1988)Phytochem. 27:2329), and cytotoxic compounds from Knema glomerate(Myristicaceae) (Zeng et al. (1994) J. Nat. Prod. 57:376).

Japanese Patent No. JP05213729A teaches the use of syntheticdihydrochalcones as melanin inhibitors for treatment of skininflammation, stains, freckles and chromatosis resulting from sun-burn.The claimed compounds have the following general formula:

wherein X is selected from H, OH or ═O; R is H or Me; and R¹-R⁵ areindependently selected from H, OR and NH₂. Thus, the discloseddihydrochalcones contain a single hydroxy/methoxy substituent on onephenyl ring and five non-specific substituents (R¹-R⁵) on the secondring. No enzyme inhibition for any of the claimed compositions wasmeasured, rather the inhibition of melanin was determined by measurementof the amount of melanin produced by cultured skin cells and colorchanges of animal skin following UV stimulation. In the currentinvention, one of the compounds disclosed in JP05213729A,1-(4-hydroxyphenyl)-3-(4′-hydroxyphenyl)-1-propanol, was synthesized andits ability to inhibit tyrosinase was measured. This compound exhibitedonly moderate inhibition of tyrosinase (IC₅₀=305 μM, Table 2.) Thepresent invention teaches novel diarylalkanes which have a uniquesubstitution pattern wherein at least one of the two aromatic rings Ar₁or Ar₂ are substituted with 1-5 R′ groups (R′₁—R′₅) and wherein at least2 of said of R′₁—R′₅ are not H). These compounds exhibit an unexpectedability to inhibit the activity of tyrosinase, which is 4-600 foldgreater than the compounds taught by JP05213729. It is believed that todate there are no published reports any of the compounds taught in theinstant application.

SUMMARY OF THE INVENTION

The present invention includes a method for inhibiting the activity ofan enzyme with a binuclear active site, referred to herein as abinuclear enzyme, said method comprising administering to a host in needthereof an effective amount of one or more diarylalkane(s), wherein saiddiarylalkanes are synthesized and/or isolated from a one or more plants.Examples of binuclear enzymes included within the scope of the presentinvention include, but are not limited to tyrosinase, arginase, urease,cytochrome c oxidase, proton pumping heme-copper oxidase, bifunctionalcarbon monoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotidereductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase,and bacterial phosphotriesterase. In one embodiment, the binuclearenzyme is tyrosinase.

The present invention also includes a method for the prevention andtreatment of diseases and conditions related to the activity ofbinuclear enzymes. The method of prevention and treatment according tothis invention comprises administering internally or topically to a hostin need thereof a therapeutically effective amount of one or morediarylalkane(s). Depending on the binuclear enzyme being inhibited thediarylalkane may be used as an anti-microbial, anti-fungal,anti-malaria, or anti-viral agent, a regulator for the production ofnitric oxide as a means of controlling male and female sexual arousal,an anti-inflammatory drug, an antioxidant, a regulator of drugmetabolism and an inhibitor or the growth of cancers and solid tumors.The diarylalkane may also be used in the prevention and treatment ofperiodontal diseases, oral pre-cancerous conditions, oral cancers, andother oral malignancies, sensitive gums and teeth, sequelae, pulpitis,irritation, pain and inflammation caused by the physical implantation oforal dentures, trauma, injuries, bruxism and other minor wounds inmouth, on the gums or on the tongue, dental plague and calculus, toothdecalcification, proteolysis and caries (decay).

The present invention further includes methods for the prevention andtreatment of diseases and conditions related to the overproduction oruneven distribution of melanin, said method comprising administeringinternally or topically to a host in need thereof a therapeuticallyeffective amount of one or more diarylalkane(s). Diseases and conditionsrelated to the overproduction or uneven distribution of melanin include,but not limited to suntan, hyper pigmentation spots caused by skinaging, liver diseases, thermal burns and topical wounds, skinpigmentation due to inflammatory conditions caused by fungal, microbialand viral infections, vitilago, carcinoma, melanoma, as well as othermammalian skin conditions.

The method can also be used for preventing and treating skin darkeningand damage resulting from exposure to ultraviolet (UV) radiation,chemicals, heat, wind and dry environments. Finally, the method can beused for preventing and treating wrinkles, saggy skin, lines and darkcircles around the eyes, soothing sensitive skin and preventing andtreating dermatitis and other allergy related conditions of the skin. Inaddition to their use for the prevention and treatment of the abovedescribed diseases and conditions of the skin, the therapeuticcompositions described herein provide an efficacious composition thatyields the benefit of smooth and youthful skin appearance with improvedskin color, enhanced elasticity, reduced and delayed aging, enhancedyouthful appearance and texture, and increased flexibility, firmness,smoothness and suppleness.

By chelating with metal ions diarylalkanes also can be used to deliveressential metal ions into the blood stream of the host, and/or carrymetal ions through the skin or blood/brain barrier, as well as, othermembranes. In this embodiment, the method comprises administering to ahost in need thereof a therapeutically effective amount of one or morediarylalkane(s), together with the metal ion(s) to be delivered. In thiscapacity the diarylalkanes can be used to treat diseases and conditionsincluding, but not limited to anemia and other iron deficiencies,inflammation; obesity and diabetes mellitus, periodontal diseases, oralpre-cancerous conditions, oral cancers, and other oral malignancies,sensitive gums and teeth, sequelae, pulpitis, irritation, pain andinflammation caused by the physical implantation of oral dentures,trauma, injuries, bruxism and other minor wounds in mouth, on the gumsor on the tongue, dental plague and calculus, tooth decalcification,proteolysis and caries (decay), viral infections insomnia, suppressedimmune function, osteoporosis, amenorrhea, dysmenorrheal, epilepsy,hypertension, cholesterolemea, coronary and cerebral vasospasms,diarrhea, Parkinson's disease, Alzheimer's disease, cancers, rheumatoidarthritis, male infertility and macular degeneration. The metal ions areselected from the group including, but not limited to copper, chromium,iron, zinc, boron, lithium, selenium, calcium, manganese, magnesiummolybdenum and other metal ions.

In yet another embodiment, the dialkylalkanes and dialkyl alkanols canbe used in the food industry to prevent browning and color changes infruits, mushrooms and other food products.

The present invention also includes a novel composition of mattercomprised of one or more diarylalkanes, wherein said diarylalkanes areselected from the group of compounds represented by the followinggeneral structure:

wherein

-   -   Ar₁ and Ar₂ are independently selected from the group consisting        of a substituted 5- or 6-membered aromatic or heteroaromatic        ring, wherein each 6-membered aromatic or heteroaromatic ring is        independently substituted with 1-5 R′ groups (R′₁—R′₅), and each        5-membered aromatic or heteroaromatic ring is substituted with        1-4 R′ groups (R′₁—R′₄), except when Ar₁ and Ar₂ are both a        6-membered aromatic ring, i.e. a phenyl group at least one of        Ar₁ and Ar₂ are substituted with 1-5 R′ groups (R′₁—R′₅),        wherein at least 2 of said of R′₁—R′₅ are not H wherein    -   R′ independently selected from the group consisting of —H, —OH,        —SH, —OR, —CN, —SR, —NH₂, —NHR, —NR₂, X, and a glycoside of a        monosaccharide or oligosaccharide comprised of 2-6        monosaccharides, wherein said monosaccharide(s) are        independently selected from the group consisting of an        aldopentose, methyl-aldopentose, aldohexose, ketohexose and        chemical derivatives thereof; wherein R is an alkyl group having        between 1-20 carbon atoms and X is a halogen, selected from the        group consisting of Cl, Br, F, I;    -   R₆, and R₇ are independently selected from the group consisting        of —H, —OH, —OR, —CN, —NHR, —NH₂ and —X, wherein R is an alkyl        group having between 1-20 carbon atoms and wherein X is a        halogen, selected from the group consisting of Cl, Br, F, I; and        n=1 to 10. In a preferred embodiment n=2-4.

The In one embodiment, said diarylalkanes are selected from the group ofcompounds represented by the following general structure:

wherein

-   -   R₁, R₂, R₃, R₄, R₅ R′₁, R′₂, R′₃, R′₄, and R′₅ are independently        selected from the group consisting of —H, —OH, —SH, —OR, —CN,        —SR, —NH₂, —NHR, —NR₂, X, and a glycoside of a monosaccharide or        oligosaccharide comprised of 2-6 monosaccharides, wherein said        monosaccharide(s) are independently selected from the group        consisting of an aldopentose, methyl-aldopentose, aldohexose,        ketohexose and chemical derivatives thereof; wherein R is an        alkyl group having between 1-20 carbon atoms and X is a halogen,        selected from the group consisting of Cl, Br, F, I, and wherein        at least 2 of R₁-R₅ or at least 2 of R′₁—R′₅ are not H;    -   R₆, and R₇ are independently selected from the group consisting        of —H, —OH, —OR, —CN, —NHR, —NH₂ and —X, wherein R is an alkyl        group having between 1-20 carbon atoms and wherein X is a        halogen, selected from the group consisting of Cl, Br, F, I; and    -   n=1 to 10. In a preferred embodiment n=2-4.

In one embodiment, the diarylalkanes of this invention are isolated fromone or more plants selected from the family of plants including, but notlimited to Compositae, Fabaceae, Lauraceae, Leguminosae, Liliaceae,Loranthaceae, Moracea, and Myristicaceae families. The diarylalkanes ofthis invention can also be extracted, concentrated, and purified fromthe genera of high plants, including but not limited to Acacia,Broussonetia, Dianella, Helichrysum, Iryanthera, Knema, Lindera,Pterocarpus, Viscum, and Xanthocercis. The diarylalkanes can be found indifferent parts of plants, including but not limited to stems, stembarks, heart woods, trunks, trunk barks, twigs, tubers, roots, rootbarks, young shoots, seeds, rhizomes, flowers and other reproductiveorgans, leaves and other aerial parts. In a preferred embodiment, thediarylalkanes are isolated from a plant or plants in the Broussonetia,Dianella, and Iryanthera genus.

In another embodiment, the diarylalkanes of this invention are obtainedby synthetic methods. Included in this invention is a method ofsynthesizing diarylalkanes and diarylalkanols said method comprisingreducing a compound having the following general structure:

wherein

-   -   R₁-R₅ and R′₁—R′₅ and n are as defined above and wherein R₆ and        R₇ together form one or more carbonyl group(s). The reducing        agent can be selected from any known reducing agent for the        reduction of ketones to alcohols including, but not limited to        borohydrides, H₂ in the presence of a catalyst, NaH and LiAlH₄.        In one embodiment the reducing agent is NaBH₄.

In yet another embodiment, the diarylalkanes are obtained by syntheticmodification of a naturally occurring compound isolated from a plantsource. For example, the naturally occurring compound butein is isolatedfrom a plant source, dehydrated and reduced to yield the correspondingdiarylalkanol.

In yet another embodiment, the diarylalkanes are obtained by thereaction of two appropriately substituted aromatic compounds. Feasiblechemical reactions for synthesizing these compounds from two substitutedaromatic compounds include, but are not limited to Aldol condensationbetween a substituted benzaldehyde and a substituted acetophenone;Claisen-Schmidt reaction or crossed aldol condensation between analdehyde and a ketone; Grignard reaction using an organomagnesium halideof one substituted aromatic ring to link the second substituted aromaticring through addition reaction to the carbonyl group on the molecule;Claisen rearrangement by an intra-molecular isomerization, in which anesterified phenol with appropriate substitution groups will beisomerized to link the second aromatic rings at the ortho-position ofthe phenol followed by a reducing reaction; and a Suzuki couplingreaction, in which two substituted aromatic rings are converted toarylboronic acids and then linked by an alkyl halide by using acarefully selected palladium catalyst. These reactions are well known tothose of skill in the art and the conditions for such reactions can bedetermined using the information disclosed herein for the synthesis ofthese compounds.

The present invention implements a strategy that combines an inhibitionassay with a chemical dereplication process to identify active plantextracts and the particular compounds within those extracts thatspecifically inhibit binuclear enzymes. This approach involves acombination of natural product isolation, organic synthesis, molecularmodeling and enzymatic inhibition assays to optimize the structure andmaximize effectiveness of the drug. This method is described in U.S.application Ser. No. 10/185,758, filed Jun. 27, 2002, entitled “Methodfor Generating, Screening and Dereplicating Natural Product Librariesfor the Discovery of Therapeutic Agents,” which is incorporated hereinby reference in its entirety. The efficacy of this method isdemonstrated using a tyrosinase inhibition assay as described in theExample section below. The purity of the diarylalkanes evaluatedaccording to the method of this invention is in the range of 0.01% to100%, depending on the methodology used to obtain the compound(s).

In a preferred embodiment, the dose of the diarylalkane administered tothe host in need thereof is an efficacious, nontoxic quantity generallyselected from the range of 0.001% to 100% based on total weight of thefinal formulation, and/or 0.01 mg to 200 mg per kilogram based on thebody weight of the host. Persons skilled in the art using routineclinical testing are able to determine optimum doses for the particularailment being treated. The present invention provides commerciallyviable options for the synthesis, and/or isolation, purification andformulation of diarylalkanes to yield composition of matter havingdesirable physiological activity. The compositions of this invention canbe administered by any method known to one of ordinary skill in the art.The modes of administration include, but are not limited to, enteral(oral) administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application. The method oftreatment according to this invention comprises administering internallyor topically to a patient in need thereof a therapeutically effectiveamount of a pure or a mixture of diarylalkanes synthesized and/orisolated from a single plant or multiple plants. In a preferredembodiment the composition is administered topically.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the process of melanogenesis, together with variouspotential mechanisms for regulating this process.

FIG. 2 illustrates the structure of the active site of tyrosinase. Ascan be seen in this figure, two copper ions are closely spaced and eachis coordinated to three histidines through the N-E nitrogen atom of itsside chain.

FIGS. 3A and 3B depict the structure, name, mechanism of action, andother effects of known tyrosinase inhibitors.

FIG. 4 illustrates the HPLC/UV chromatogram of a HTP fraction thatcontains the UP288(1-(2-methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane)(1) as highlighted.

FIG. 5 depicts the chemical structure and ¹³C-NMR spectrum of UP288 (1).

FIG. 6 illustrates tyrosinase inhibitory dose response curves and IC₅₀values for UP288 and kojic acid.

FIG. 7 depicts the bioassay-guided isolation of two active compounds(UP302a and UP302b) from Dianella ensifolia (P0389) (whole plant).

FIG. 8 depicts the HPLC/UV chromatogram of the enriched UP302 fractionafter multiple column separations.

FIG. 9 depicts a gHSQC spectrum of UP302a (2), which illustrates thelinks between protons and carbons.

FIG. 10 illustrates graphically inhibition of the activity of tyrosinaseat various concentrations of inhibitor UP302a and substrate L-DOPA. Thiskinetic study revealed that UP302a is a competitive inhibitor of thetyrosinase enzyme.

FIG. 11 illustrates the inhibition of endogeneous melanin productionfrom mouse B16 F1 cells by kojic acid and UP302a (2). Each sample wastested in triplicate at 10 different concentrations.

FIG. 12 depicts three-dimensional conformation of UP302a after MM2energy minimization.

FIG. 13 illustrates the three-dimensional conformation of UP302a, whencoordinated to two copper ions in the Cu^(II)—O₂—Cu^(II) oxidationstate.

FIG. 14 depicts the distances between adjacent atoms of UP302a whenchelated with copper ions in the peroxide form (Cu^(II)—O₂—Cu^(II)) ascalculated in Example 17.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates generally to the prevention and treatmentof diseases and conditions mediated by binuclear enzymes. Specifically,the present invention includes a method for inhibiting the activity ofan enzyme having a binuclear active site. Included in the presentinvention are novel compositions comprised of one or morediarylalkane(s). The diarylalkanes of the present invention can beisolated from one or more plant sources or can be obtained by organicsynthesis. Further included in the present invention are methods forisolating these compounds from a natural source and methods forsynthesizing these compounds. In one embodiment, the diarylalkanes areobtained by synthetic modification of a naturally occurring compoundisolated from a plant source.

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.Unless defined otherwise all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs.

It is to be noted that as used herein the term “a” or “an” entity refersto one or more of that entity; for example, a diarylalkane refers to oneor more diarylalkanes. As such, the terms “a” or “an”, “one or more” and“at least one” are used interchangeably herein.

“Diarylalkanes” as used herein are a specific class of aromaticcompounds having the following general structure: The present inventionalso includes a novel composition of matter comprised of one or morediarylalkanes, wherein said diarylalkanes are selected from the group ofcompounds represented by the following general structure:

wherein

-   -   Ar₁ and Ar₂ are independently selected from the group consisting        of a substituted 5- or 6-membered aromatic or heteroaromatic        ring, wherein each 6-membered aromatic or heteroaromatic ring is        independently substituted with 1-5 R′ groups (R′₁—R′₅), and each        5-membered aromatic or heteroaromatic ring is substituted with        1-4 R′ groups (R′₁—R′₄), except when Ar₁ and Ar₂ are both a        6-membered aromatic ring, i.e. a phenyl group at least one of        Ar₁ and Ar₂ are substituted with 1-5 R′ groups (R′₁—R′₅),        wherein at least 2 of said of R′₁—R′₅ are not H wherein    -   R′ is independently selected from the group consisting of —H,        —OH, —SH, —OR, —CN, —SR, —NH₂, —NHR, —NR₂ and X, and a glycoside        of a monosaccharide or oligosaccharide comprised of 2-6        monosaccharides, wherein said monosaccharide(s) are        independently selected from the group consisting of an        aldopentose, methyl-aldopentose, aldohexose, ketohexose and        chemical derivatives thereof; wherein R is an alkyl group having        between 1-20 carbon atoms and X is a halogen, selected from the        group consisting of Cl, Br, F and I;    -   R₆, and R₇ are independently selected from the group consisting        of —H, —OH, —OR, —CN, —NHR, —NH₂, and —X, wherein R is an alkyl        group having between 1-20 carbon atoms and wherein X is a        halogen, selected from the group consisting of Cl, Br, F, I; and        n=1 to 10. In a preferred embodiment n=2-4.

In one embodiment, said diarylalkanes and diarylalkanols are selectedfrom the group of compounds represented by the following generalstructure:

wherein

-   -   R₁, R₂, R₃, R₄, R₅ R′₁, R′₂, R′₃, R′₄, and R′₅ are independently        selected from the group consisting of —H, —OH, —SH, —OR, —CN,        —SR, —NH₂, —NHR, —NR₂, X, and a glycoside of a monosaccharide or        oligosaccharide comprised of 2-6 monosaccharides, wherein said        monosaccharide(s) are independently selected from the group        consisting of an aldopentose, methyl-aldopentose, aldohexose,        ketohexose and chemical derivatives thereof; wherein R is an        alkyl group having between 1-20 carbon atoms and X is a halogen,        selected from the group consisting of Cl, Br, F, I, and wherein        at least 2 of R₁-R₅ or at least 2 of R′₁—R′₅ are not H;    -   R₆, and R₇ are independently selected from the group consisting        of —H, —OH, —OR, —CN, —NHR, —NH₂, and —X, wherein R is an alkyl        group having between 1-20 carbon atoms and wherein X is a        halogen, selected from the group consisting of Cl, Br, F and I;        and    -   n=1 to 10. In a preferred embodiment n=2-4.

“Diarylalkanols” as used herein are a specific type of “diarylalkanes”having at least one hydroxyl group (R₆ and/or R₇=−OH) attached to thealkyl carbons between the two aromatic rings.

“Binuclear enzyme” as used herein refers to an enzyme which has abinuclear active site, an example of which is tyrosinase which has twocopper ions at its active site as discussed above. Binuclear enzymesinclude, but are not limited to tyrosinase, arginase, urease, cytochromec oxidase, proton pumping heme-copper oxidase, bifunctional carbonmonoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotidereductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase,and bacterial phosphotriesterase.

“Therapeutic” as used herein, includes prevention, treatment and/orprophylaxis. When used, therapeutic refers to humans as well as otheranimals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the alleviation of the signs, symptoms or causes of adisease or any other alteration of a biological system that is desired.The precise dosage will vary according to a variety of factors,including but not limited to the age and size of the subject, thedisease and the treatment being effected.

“Placebo” refers to the substitution of the pharmaceutically ortherapeutically effective dose or amount dose sufficient to induce adesired biological that may alleviate the signs, symptoms or causes of adisease with a non-active substance.

A “host” or “patient” or “subject” is a living mammal, human or animal,for whom therapy is desired. The “host,” “patient” or “subject”generally refers to the recipient of the therapy to be practicedaccording to the method of the invention. It should be noted that theinvention described herein may be used for veterinary as well as humanapplications and that the term “host” should not be construed in alimiting manner. In the case of veterinary applications, the dosageranges can be determined as described below, taking into account thebody weight of the animal.

As used herein a “pharmaceutically acceptable carrier” refers to anycarrier, which does not interfere with effectiveness of the biologicalactivity of the active ingredient and which is not toxic to the host towhich it is administered. Examples of “pharmaceutically acceptablecarriers” include, but are not limited to, any of the standardpharmaceutical carriers such as a saline solution, i.e. Ringer'ssolution, a buffered saline solution, water, a dextrose solution, serumalbumin, and other excipients and preservatives for tableting andcapsulating formulations.

The present invention includes a method for inhibiting the activity ofan enzyme with a binuclear active site, referred to herein as abinuclear enzyme, said method comprising administering to a host in needthereof an effective amount of one or more diarylalkane(s), wherein saiddiarylalkanes are synthesized and/or isolated from a one or more plants.Examples of binuclear enzymes included within the scope of the presentinvention include, but are not limited to tyrosinase, arginase, urease,cytochrome c oxidase, proton pumping heme-copper oxidase, bifunctionalcarbon monoxide dehydrogenase/acetyl-coenzyme A synthase, ribonucleotidereductase, metalo-beta-lactamase, H(+)-ATPase and alternative oxidase,and bacterial phosphotriesterase. In one embodiment, the binuclearenzyme is tyrosinase.

The present invention also includes a method for the prevention andtreatment of diseases and conditions related to the activity ofbinuclear enzymes. The method of prevention and treatment according tothis invention comprises administering internally or topically to a hostin need thereof a therapeutically effective amount of one or morediarylalkane(s). Depending on the binuclear enzyme being inhibited thediarylalkane may be used as an anti-microbial, anti-fungal,anti-malaria, or anti-viral agent, a regulator for the production ofnitric oxide as a means of controlling male and female sexual arousal,an anti-inflammatory drug, an antioxidant, a regulator of drugmetabolism, for treatment and prevention of periodontal diseases, oralpre-cancerous conditions, oral cancers, and other oral malignancies,sensitive gums and teeth, sequelae, pulpitis, irritation, pain andinflammation caused by the physical implantation of oral dentures,trauma, injuries, bruxism and other minor wounds in mouth, on the gumsor on the tongue, dental plague and calculus, tooth decalcification,proteolysis and caries (decay). and an inhibitor of the growth ofcancers and solid tumors.

The present invention further includes methods for the prevention andtreatment of diseases and conditions related to the overproduction oruneven distribution of melanin, said method comprising administeringinternally or topically to a host in need thereof a therapeuticallyeffective amount of one or more diarylalkane(s). Diseases and conditionsrelated to the overproduction or uneven distribution of melanin include,but not limited to suntan, hyper pigmentation spots caused by skinaging, liver diseases, thermal burns and topical wounds, skinpigmentation due to inflammatory conditions caused by fungal, microbialand viral infections, vitilago, carcinoma, melanoma, as well as othermammalian skin conditions.

The method can also be used for preventing and treating skin darkeningand damage resulting from exposure to ultraviolet (UV) radiation,chemicals, heat, wind and dry environments. Finally, the method can beused for preventing and treating wrinkles, saggy skin, lines and darkcircles around the eyes, soothing sensitive skin and preventing andtreating dermatitis and other allergy related conditions of the skin. Inaddition to their use for the prevention and treatment of the abovedescribed diseases and conditions of the skin, the therapeuticcompositions described herein provide an efficacious composition thatyields the benefit of smooth and youthful skin appearance with improvedskin color, enhanced elasticity, reduced and delayed aging, enhancedyouthful appearance and texture, and increased flexibility, firmness,smoothness and suppleness.

By chelating with metal ions diarylalkanes also can be used to deliveressential metal ions into the blood stream of the host, and/or carrymetal ions through the skin or blood/brain barrier, as well as, othermembranes. In this embodiment, the method comprises administering to ahost in need thereof a therapeutically effective amount of one or morediarylalkane(s), together with the metal ion(s) to be delivered. In thiscapacity the diarylalkanes can be used to treat diseases and conditionsincluding, but not limited to anemia and other iron deficiencies,inflammation; obesity and diabetes, periodontal diseases, oralpre-cancerous conditions, oral cancers, and other oral malignancies,sensitive gums and teeth, sequelae, pulpitis, irritation, pain andinflammation caused by the physical implantation of oral dentures,trauma, injuries, bruxism and other minor wounds in mouth, on the gumsor on the tongue, dental plague and calculus, tooth decalcification,proteolysis and caries (decay), and viral infections. The metal ions areselected from the group including, but not limited to copper, iron,zinc, selenium, magnesium and other metal ions.

In yet another embodiment, the dialkylalkanes can be used in the foodindustry to prevent browning and color changes in fruits, mushrooms andother food products.

The diarylalkanes that can be used in accordance with the followinginclude compounds illustrated by the general structure set forth above.The diarylalkanes of this invention may be obtained by synthetic methodsor may be isolated from one or more families of plants selected from thegroup including, but not limited to Compositae, Fabaceae, Lauraceae,Leguminosae, Liliaceae, Loranthaceae, Moracea, and Myristicaceae. Thediarylalkanes of this invention can be extracted, concentrated, andpurified from the genera of high plants, including but not limited toAcacia, Broussonetia, Dianella, Helichrysum, Iryanthera, Knema, Lindera,Pterocarpus, Viscum, and Xanthocercis. The diarylalkanes can be found indifferent parts of the plant, including but not limited to stems, stembarks, heart woods, trunks, trunk barks, twigs, tubers, roots, rootbarks, young shoots, seeds, rhizomes, flowers and other reproductiveorgans, leaves and other aerial parts. In a one embodiment, thediarylalkanes are isolated from a plant or plants in the Broussonetia,Dianella, and Iryanthera genera.

In another embodiment, the diarylalkanes of this invention are obtainedby synthetic methods. Included in this invention is a method ofsynthesizing diarylalkanes and diarylalkanols said method comprisingreducing a compound having the following general structure:

wherein

-   -   R₁-R₅ and R′₁—R′₅ and n are as defined above and wherein R₆ and        R₇ together form one or more carbonyl group(s). The reducing        agent can be selected from any known reducing agent for the        reduction of ketones to alcohols including, but not limited to        borohydrides, H₂ in the presence of a catalyst, NaH and LiAlH₄.        In one embodiment the reducing agent is NaBH₄.

In yet another embodiment, the diarylalkanes are obtained by syntheticmodification of a naturally occurring compound isolated from a plantsource. For example, the naturally occurring compound butein is isolatedfrom a plant source, dehydrated and reduced to yield the correspondingdiarylalkanol.

In yet another embodiment, the diarylalkanes are obtained by thereaction of two appropriately substituted aromatic compounds. Feasiblechemical reactions for synthesizing these compounds from two substitutedaromatic compounds include, but are not limited to Aldol condensationbetween a substituted benzaldehyde and a substituted acetophenone;Claisen-Schmidt reaction or crossed aldol condensation between analdehyde and a ketone; Grignard reaction using an organomagnesium halideof one substituted aromatic ring to link the second substituted aromaticring through addition reaction to the carbonyl group on the molecule;Claisen rearrangement by an intra-molecular isomerization, in which anesterified phenol with appropriate substitution groups will beisomerized to link the second aromatic rings at the ortho-position ofthe phenol followed by a reducing reaction; and a Suzuki couplingreaction, in which two substituted aromatic rings are converted toarylboronic acids and then linked by an alkyl halide by using acarefully selected palladium catalyst. These reactions are well known tothose of skill in the art and the conditions for such reactions can bedetermined using the information disclosed herein for the synthesis ofthese compounds.

Note that throughout this application various citations are provided.Each of these citations is specifically incorporated herein by referencein its entirety.

The present invention implements a strategy that combines a tyrosinaseinhibition assay with a chemical dereplication process to identifyactive plant extracts and the particular compounds within those extractsthat specifically inhibit the binuclear enzyme tyrosinase. As notedabove, enzymes that inhibit tyrosinase may lead to a reduction in theproduction of melanin thereby effectively lightening the skin. A libraryof plant extracts was generated by extracting dry plant powders with anorganic solvent, as described in Example 1. The tyrosinase inhibitionassay was developed following a method reported by Jones et al. (2002)Pigment. Cell Res. 15:335, as described in Example 2. Using this assay,a total of 1144 plant extracts were screened for their ability toinhibit the activity of mushroom tyrosinase. This primary screenidentified 20 plant extracts (1.75% hit rate) with potent tyrosinaseinhibitory activity. Table 1 delineates percent inhibition of tyrosinaseby four of these extracts isolated from four different genera.

In order to efficiently identify active compounds from the active plantextracts, a high throughput fractionation process was used, as describedin Example 3. Briefly, the active extracts were fractionated using ahigh throughput purification (HTP) system. Each of the fractions wasthen tested for its ability to inhibit tyrosinase activity as per theprimary assay described in Example 2. After dereplication, using acombination of HPLC with PDA and MS detectors coupled with a structuredatabase search and elimination of fractions that contained knowntyrosinase inhibitors, such as polyphenols and chromones, a total ofseven active extracts were chosen for bioassay guided large-scaleisolation and purification as described in Examples 4-6, using theextracts of Broussonetia kazinoki Sieb. Et Zucc (Moraceae), and Dianellaensifolia (L.) DC. (Liliaceae) for purposes of illustration.

Example 4 describes the extraction, separation and purification of thenovel diarylpropane:1-(2-methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane(UP288) (1) from Broussonetia kazinoki Sieb. Et Zucc (Moraceae) (wholeplant) using the general method set forth in Examples 1-3. FIG. 4illustrates the HPLC/UV chromatogram of a HTP fraction that contains theUP288. The structure of the active compound UP288 was elucidated using acombination of spectroscopic methods as set forth in Example 4. FIG. 5depicts the chemical structure and ¹³C-NMR spectrum of UP288. FIG. 6illustrates tyrosinase inhibitory dose response curves and IC₅₀ valuesfor UP288 relative to kojic acid. The figure illustrates that UP288 (1)is as potent a tyrosinase inhibitor as kojic acid, having an IC₅₀=24 μM.

Surprisingly, two similar diarylalkanes were isolated and identifiedfrom a totally different family of plant—Dianella ensifolia (L.) DC.(Liliaceae), as described in Example 5. FIG. 7 depicts schematically thebioassay-guided isolation of these two active compounds (UP302a (2) andUP302b (3)) from Dianella ensifolia (P0389) (whole plant). Withreference to FIG. 7 , it can be seen that only fifteen column fractionsfrom a total of 264 collected samples exhibited potent inhibition oftyrosinase. A HPLC analysis (FIG. 8 ) of the combined active fractionsshowed that active compounds were minor components in the best pool,which has already been heavily enriched. Laborious separation andpurification efforts yielded two novel active compounds that have beenfully characterized by NMR and other spectroscopic methods asillustrated in Example 5 and FIG. 9 as1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane(UP302a, IC₅₀=0.24 μM) (2) and1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,5′-dihydroxyphenyl)-propane(IP302b, IC₅₀=1.2 μM) (3).

Example 6 describes the large-scale isolation of UP302a (2), the mostpotent tyrosinase inhibitor, isolated from Dianella ensifolia (DE)(whole plant). With reference to Example 6, from 4.3 kg of driedbiomass, a total of 30 mg of pure UP302a (2) was obtained after multiplecolumn fractionations on silica gel, CG-161, and C-18 resins. Thestructure and biological function of the isolated compound wereconfirmed.

Due to the low natural abundance of diarylalkanes/diarylalkanols methodsto synthesize these biologically active compounds as an alternativecommercial source of this class of compounds was developed. Example 7describes a general method for the synthesis of diarylalkanes via thereduction of substituted chalcones. For purposes of illustration thereduction of 2,4-dihydroxyphenyl)-3′,4′-dimethoxyphenylchalcone (4) to1-(2,4-dihydroxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol (5) usingsodium borohydride is described. However, as set forth in Example 7, anumber of other diarylalkanes have been synthesized using this generalmethod. All of the compounds synthesized showed high to moderatetyrosinase inhibitory activity. With respect to the general methoddescribed in Example 7, any other known reducing agents, can be used toeffect this reduction, including, but are not limited to otherborohydrides, H₂ in the presence of a catalyst, NaH and LiAlH₄.

Using the general reaction described in Example 7, several othersubstituted diarylpropanones have been converted to diarylpropanesand/or diarylpropanols as demonstrated in Examples 8, 9 and 10. Example11 demonstrates the synthesis of a diarylpropanol using a flavonoidglycoside isolated from a natural source as the starting material.

In another embodiment, the present invention includes methods forsynthesizing this class of compounds by reaction of two appropriatelysubstituted aromatic compounds. This embodiment is illustrated inExample 12, using the reaction of resorcinol with3-methoxy-4-hydroxycinnamic acid for purposes of illustration. Feasiblechemical reactions for synthesizing these compounds from two substitutedaromatic compounds include, but are not limited to Aldol condensationbetween a substituted benzaldehyde and a substituted acetophenone;Claisen-Schmidt reaction or crossed aldol condensation between analdehyde and a ketone; Grignard reaction using an organomagnesium halideof one substituted aromatic ring to link the second substituted aromaticring through addition reaction to the carbonyl group on the molecule;Claisen rearrangement by an intra-molecular isomerization, in which anesterified phenol with appropriate substitution groups will beisomerized to link the second aromatic rings at the ortho-position ofthe phenol followed by a reducing reaction; and a Suzuki couplingreaction, in which two substituted aromatic rings are converted toarylboronic acids and then linked by an alkyl halide by using acarefully selected palladium catalyst. These reactions are well known tothose of skill in the art and the conditions for such reactions can bedetermined using the information disclosed herein for the synthesis ofthese compounds.

Example 13 sets forth the IC₅₀ values for a number of diarylalkanes anddiarylalkanols synthesized according the methods of this invention. Thecompounds were evaluated using the general method described in Example2. The IC₅₀ value of each sample was calculated using kinetics softwareto verify that the reaction was linear at a specified time andconcentration. Using the methods described in Examples 7-12a total of 24compounds were synthesized and evaluated for their ability to inhibittyrosinase. The results are set forth in Table 2. With reference toTable 2, it can be seen that the IC₅₀'s of the synthetic diarylalkanolswere comparable to the naturally occurring diarylpropanes. Thus, thesetwo classes of compounds are capable of inhibiting tyrosinase toapproximately the same extent. The most active diarylalkanes and/ordiarylalkanols had three carbons between the two aromatic rings. Usingthe calculations described in Example 17, this structural feature wasdemonstrated to be critical in order to generate a parallel andsuperimposed intra-molecular conformations. However, diarylalkanols,which contained two and four carbons between the two aromatic rings,such as 1-(2,4-dihydroxyphenyl)-2-(4′-methoxyphenyl)-1-ethanol (IC₅₀=77μM) and 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethyl-buthane (IC₅₀=700 μM)also were able to significantly inhibit tyrosinase activity.

Using the method described in Example 2, the inhibition of tryosinase byUP302a (2) was evaluated using L-DOPA as the substrate as set forth inExample 14. The results are set forth in FIG. 10 . This study revealedthat UP302a (2) is a powerful competitive inhibitor having a longlasting effect. Interestingly, tyrosinase activity was not resumed forseveral days after incubation with UP302a. In contrast, tyrosinaseactivity was totally restored after only 1 hour following incubationwith kojic acid. Since two of the substituents on the aromatic rings ofUP302a were methoxy groups, the inhibitor cannot be easily hydroxylatedand/or oxidized. This may explain both the effectiveness and duration ofthe inhibitory activity of UP302a. Thus, it can be concluded that thesecompounds will have a long lasting effect.

The efficacy of the claimed composition was also demonstrated bymeasuring the inhibition of melanin produced in an in vitro test on aB-16 cell line as described in Example 15. The results are set forth inFIG. 11 . The reduction of endogenous melanin by UP302a (2) was almostsix fold greater than that of kojic acid. Additionally, inhibition ofMSH induced melanin production by UP302a was also significantly greaterthan kojic acid. As expected, UP288 (1) was comparable to kojic acid inthe B-16 cell line model.

Example 16 describes an assay to assess the cytotoxicity of twodiarylpropanes UP288 (1) and UP302a (2) relative to kojic acid. At aconcentration of 250 μM, which was above IC₅₀ of all three testedcompounds, the diarylpropanes demonstrated similar safety profiles tothat of kojic acid.

Example 17 describes the molecular modeling analyses performed todetermine the most stable 3-D conformation of the active diarylalkanesand diarylalkanols. Molecular mechanics calculations were performedusing Chem3D software. These calculations revealed that the most potenttyrosinaseinhibitor—1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane(UP302a (2), IC₅₀=0.24 μM) has a very unique 3-dimensional conformationwith two the aromatic rings superimposed on each other as illustrated inFIG. 12 . The minimized total energy for the conformation is −4.7034KJ/Mol and the distance between the two aromatic rings is 3.28 Å. Thephenolic hydroxyl groups on the first aromatic ring are right above thetwo methoxy groups on the second aromatic ring with the distance betweentwo oxygen atoms being as 2.99 Å and 3.16 Å, respectively as illustratedin FIG. 14 . The active site of the binuclear enzyme tyrosinase has twocopper ions complexed to an oxygen molecule in a peroxide oxidationstate [Cu^(II)—O₂—Cu^(II)], which is key to the mechanism by whichtyrosinase catalyzes the introduction of a hydroxyl group in the orthoposition of the aromatic ring of a mono-phenol (such as tyrosine).(Decker et al. (2000) Angew. Chem. Int. Ed. 39:1591). The interactomicdistances were reported as 3.56 Å for Cu—Cu. 1.90 Å for Cu—O and 1.41 Åfor 0-0. (Kitajima et al. (1989) J. Am. Chem. Soc. 111:8975). Theparallel conformation of1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane(UP302a, IC₅₀=0.24 μM) will perfectly chelate with both copper ions ofthe [Cu^(II)—O₂—Cu^(II)] complex from both the top and the bottom asillustrated in FIGS. 13 and 14 . This dual chelation by the inhibitor toboth copper ions at the active site will totally block the access of thesubstrate, such as L-Dopa to the enzyme, thus effectively inhibiting thefunction of the protein. Using the same approach, the isolated andsynthetic diarylalkanes and diarylalkanols listed in Table 2 wereanalyzed. The results of this analysis indicated that the compounds withtwisted or non-parallel conformations possessed either no ability oronly a weak ability to inhibit the activity of tyrosinase.

From these studies it has been determined that the most effectivediarylalkane inhibitors have two to three substituents on one aromaticring and zero to multiple substituents on the second aromatic ring. Themost favorable structures are those in which at least one aromatic ringis substituted in the 2 and 4-positions. Preferably the rings are6-membered aromatic and/or heteroaromatic as demonstrated by two of thecompounds isolated1-(2-hydroxy-4-methoxyphenyl)-3-(2′,3′,4′,5′-tetrahydro-bezo(b)dioxocin-8-yl)-1-propanol—IC₅₀=72μM and3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxyphenyl)-1-propanol—IC₅₀=225μM.

The compositions of this invention can be administered by any methodknown to one of ordinary skill in the art. The modes of administrationinclude, but are not limited to, enteral (oral) administration,parenteral (intravenous, subcutaneous, and intramuscular) administrationand topical application. The method of treatment according to thisinvention comprises administering internally or topically to a patientin need thereof a therapeutically effective amount of a diarylalkane ora mixture comprised of two or more diarylalkanes.

The compositions of the present invention can be formulated aspharmaceutical compositions, which include other components such as apharmaceutically and/or cosmetically acceptable excipient, an adjuvant,and/or a carrier. For example, compositions of the present invention canbe formulated in an excipient that the host to be treated can tolerate.An excipient is an inert substance used as a diluent or vehicle for atherapeutic agent such as a diarylalkane or a mixture of diarylalkanes.Examples of such excipients include, but are not limited to water,buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank'ssolution, preservatives and other aqueous physiologically balanced saltsolutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyloleate, or triglycerides may also be used. Other useful formulationsinclude suspensions containing viscosity-enhancing agents, such assodium carboxymethylcellulose, sorbitol or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer, tris buffer, histidine, citrate,and glycine, or mixtures thereof, while examples of preservativesinclude, but are not limited to EDTA, disodium EDTA, BHA, BHT, vitaminC, vitamin E, sodium bisulfite, SnCl₂, thimerosal, m- or o-cresol,formalin and benzyl alcohol. Standard formulations can be either liquidor solids, which can be taken up in a suitable liquid as a suspension orsolution for administration. Thus, in a non-liquid formulation, theexcipient can comprise dextrose, human serum albumin, preservatives,etc., to which sterile water or saline can be added prior toadministration.

In one embodiment of the present invention, the composition can alsoinclude an adjuvant or a carrier. Adjuvants are typically substancesthat generally enhance the biological response of a host to a specificbioactive agent. Suitable adjuvants include, but are not limited to,Freund's adjuvant, other bacterial cell wall components, aluminum,magnesium, copper, zinc, iron, calcium, and other metal ion based salts,silica, polynucleotides, toxoids, serum proteins, viral coat proteins,other bacterial-derived preparations, gamma interferon; block copolymeradjuvants; such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.), Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated host. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, viruses, oils, esters, and glycols.

In one embodiment, the composition is prepared as a controlled releaseformulation, which slowly releases the composition of the presentinvention into the host. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehicles will beknown to those skilled in the art. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

The therapeutic agents of the instant invention are preferablyadministered topically by any suitable means, known to those of skill inthe art for topically administering therapeutic compositions including,but not limited to as an ointment, gel, lotion, or cream base, or as atoothpaste, mouth-wash, or coated on dental flossing materials or as anemulsion, as a patch, dressing or mask, a nonsticking gauze, a bandage,a swab or a cloth wipe. Example 18 describes the preparation of twocream formulations with an active content at 0.01% and 0.1% of a pureand/or mixture of diarylalkane(s) in the total weight of the formula.Such topical application can be locally administered to any affectedarea, using any standard means known for topical administration. Atherapeutic composition can be administered in a variety of unit dosageforms depending upon the method of administration. For particular modesof delivery, a therapeutic composition of the present invention can beformulated in an excipient of the present invention. A therapeuticreagent of the present invention can be administered to any host,preferably to mammals, and more preferably to humans. The particularmode of administration will depend on the condition to be treated.

In one embodiment, a suitable ointment is comprised of the desiredconcentration of a single diarylalkane or a mixture of two or morediarylalkanes, that is an efficacious, nontoxic quantity generallyselected from the range of 0.001% to 100% based on the total weight ofthe topical formulation, from 65 to 100% (preferably 75 to 96%) of whitesoft paraffin, from 0 to 15% of liquid paraffin, and from 0 to 7%(preferably 3 to 7%) of lanolin or a derivative or synthetic equivalentthereof. In another embodiment the ointment may comprise apolyethylene-liquid paraffin matrix.

In one embodiment, a suitable cream is comprised of an emulsifyingsystem together with the desired concentration of a single diarylalkaneor a mixture of two or more diarylalkanes as provided above. Theemulsifying system is preferably comprised of from 2 to 10% ofpolyoxyethylene alcohols (e.g. the mixture available under the trademarkCetomacrogol™ 1000), from 10 to 25% of stearyl alcohol, from 20 to 60%of liquid paraffin, and from 10 to 65% of water; together with one ormore preservatives, for example from 0.1 to 1% ofN,N″-methylenebis[N′-[3-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea](available under the name Imidurea USNF), from 0.1 to 1% of alkyl4-hydroxybenzoates (for example the mixture available from NipaLaboratories under the trade mark Nipastat), from 0.01 to 0.1% of sodiumbutyl 4-hydroxybenzoate (available from Nipa Laboratories under thetrade mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol.

In one embodiment, a suitable gel is comprised of a semi-solid system inwhich a liquid phase is constrained within a three dimensional polymericmatrix with a high degree of cross-linking The liquid phase may becomprised of water, together with the desired amount of a singlediarylalkane or a mixture of two or more diarylalkanes, from 0 to 20% ofwater-miscible additives, for example glycerol, polyethylene glycol, orpropylene glycol, and from 0.1 to 10%, preferably from 0.5 to 2%, of athickening agent, which may be a natural product, selected from thegroup including, but not limited to tragacanth, pectin, carrageen, agarand alginic acid, or a synthetic or semi-synthetic compound, selectedfrom the group including, but not limited to methylcellulose andcarboxypolymethylene (carbopol); together with one or morepreservatives, selected from the group including, but not limited to forexample from 0.1 to 2% of methyl 4-hydroxybenzoate (methyl paraben) orphenoxyethanol-differential. Another suitable base, is comprised of thedesired amount of a single diarylalkane or a mixture of diarylalkanes,together with from 70 to 90% of polyethylene glycol (for example,polyethylene glycol ointment containing 40% of polyethylene glycol 3350and 60% of polyethylene glycol 400, prepared in accordance with the U.S.National Formulary (USNF)), from 5 to 20% of water, from 0.02 to 0.25%of an anti-oxidant (for example butylated hydroxytoluene), and from0.005 to 0.1% of a chelating agent (for example ethylenediaminetetraacetic acid (EDTA)).

The term soft paraffin as used above encompasses the cream or ointmentbases white soft paraffin and yellow soft paraffin. The term lanolinencompasses native wool fat and purified wool fat. Derivatives oflanolin include in particular lanolins which have been chemicallymodified in order to alter their physical or chemical properties andsynthetic equivalents of lanolin include in particular synthetic orsemisynthetic compounds and mixtures which are known and used in thepharmaceutical and cosmetic arts as alternatives to lanolin and may, forexample, be referred to as lanolin substitutes.

One suitable synthetic equivalent of lanolin that may be used is thematerial available under the trademark Softisan™ known as Softisan 649.Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is aglycerine ester of natural vegetable fatty acids, of isostearic acid andof adipic acid; its properties are discussed by H. Hermsdorf in Fette,Seifen, Anstrichmittel, Issue No. 84, No. 3 (1982), pp. 3-6.

The other substances mentioned hereinabove as constituents of suitableointment or cream bases and their properties are discussed in standardreference works, for example pharmacopoeia. Cetomacrogol 1000 has theformula CH₃(CH₂)_(m)(OCH₂CH₂)—OH, wherein m may be 15 or 17 and n may be20 to 24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol.Nipastat is a mixture of methyl, ethyl, propyl and butyl4-hydroxybenzoates.

The compositions of the invention may be produced by conventionalpharmaceutical techniques. Thus the aforementioned compositions, forexample, may conveniently be prepared by mixing together at an elevatedtemperature, preferably 60-70° C., the soft paraffin, liquid paraffin ifpresent, and lanolin or derivative or synthetic equivalent thereof. Themixture may then be cooled to room temperature, and, after addition ofthe hydrated crystalline calcium salt of mupirocin, together with thecorticosteroid and any other ingredients, stirred to ensure adequatedispersion.

Regardless of the manner of administration, the specific dose iscalculated according to the approximate body weight of the host. Furtherrefinement of the calculations necessary to determine the appropriatedosage for treatment involving each of the above mentioned formulationsis routinely made by those of ordinary skill in the art and is withinthe scope of tasks routinely performed by them without undueexperimentation, especially in light of the dosage information andassays disclosed herein. These dosages may be ascertained through use ofthe established assays for determining dosages utilized in conjunctionwith appropriate dose-response data.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Organic Extracts from Dry Plants

Dried plant material was ground to a particle size of no larger than 2mm and a portion (60 g) was transferred to an Erlenmeyer flask andextracted with 600 ml of methanol:dichloromethane (1:1). The mixture wasshaken for one hour, filtered and the biomass was extracted again withmethanol:dichloromethane (1:1) (600 ml). The organic extracts werecombined and evaporated under vacuum to provide an organic extract fromeach plant material. Each extract (approximately 75 mg) was thendissolved in 1.5 ml DMSO to a concentration of 50 mg/ml, which was thenstored in a −70° C. freezer. An aliquot of the extract solution was usedfor tyrosinase assay as described in Example 2.

Example 2 Tyrosinase Inhibition Assay

A tyrosinase inhibition assay was carried out using the method reportedby Jones et al. (2002) Pigment. Cell Res. 15:335. Using this method, theconversion of L-Dopa, a substrate of tyrosinase, into dopachrome isfollowed by monitoring absorption at 450 nm. Tyrosinase was prepared in50 mM potassium phosphate buffer, pH 6.8 (assay buffer) at 2000 U/ml andstored at −20° C. in 1 ml aliquots prior to use. For use in assays,stock enzyme solutions were thawed and diluted to 200 U/ml with assaybuffer. A 2 mM working solution of substrate, L-DOPA, was prepared inassay buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml)and diluted to 5 ml with assay buffer. The reaction mixture consisted of0.050 ml 2 mM L-DOPA, 0.050 ml 200 U/ml mushroom tyrosinase and 0.050 mlinhibitor. Reaction volume was adjusted to 200 μl with assay buffer.Assays were performed in 96 well Falcon 3097 flat-bottom microtiterplates (Beckton Dickinson, N.J.). Appearance of dopachrome was measuredwith a WALLAC 1420 Multilable Counter (Turku, Finland). Average velocitywas determined from linear enzyme rate as measured by change inabsorbance (ΔA₄₅₀) at 450 nm per minute. Percent inhibition oftyrosinase by test samples was determined by comparison of absorbance ofsamples versus control using formula (I):(Negative control absorption−sample absorption)/Negative controlabsorption×100  (1)

The results are set forth in Table 1.

TABLE 1 Tyrosinase inhibitory activity of four plant extracts PlantLatin Weight of Percent Inhibition Name and the Organic of TyrosinaseParts Amount Extract (concentration mg/ml) Broussonetica 20 g 1.1 g 68%kazinoki whole (at 0.125 mg/ml) plant Rhus chinensis 20 g 12.8 g 31%cecidiums (at 0.125 mg/ml) Polygonum 20 g 2.4 g 43% multiflorum (at0.125 mg/ml) tubers Dianella 20 g 1.7 g 57% ensifolia whole (at 0.125mg/ml) plant

Example 3 HTP Fractionation of Active Plant Extracts

Active organic extract (400 mg) was loaded onto a prepacked, normalphase, flash column. (2 cm ID×8.2 cm, 10 g silica gel). The column waseluted using a Hitachi high throughput purification (HTP) system with agradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from100% A to 100% B in 30 minutes at a flow rate of 5 mL/min. Theseparation was monitored using a broadband wavelength UV detector andthe fractions were collected in a 96-deep-well plate at 1.9 mL/wellusing a Gilson fraction collector. The sample plate was dried under lowvacuum and centrifugation. DMSO (1.5 mL) was used to dissolve thesamples in each cell and a portion (100 μL) was taken for the tyrosinaseinhibition assay in duplicate.

Example 4 Extraction, Separation and Purification of1-(2-Methoxy-4-Hydroxyphenyl)-3-(2′-Hydroxy-5′-Methoxyphenyl)-Propane(1) from Broussonetia kazinoki (BK) (Whole Plant)

Broussonetia kazinoki (100 g whole plant) was ground and extracted threetimes with 800 ml of MeOH:DCM (1:2). Dried extract (6 g) wasfractionated using a silica gel column with gradient solvent elution ofhexane/ethyl acetate (50/50) to MeOH. Fractions were collected in 2 setsof 88 test tubes. LC/MS/PDA was utilized to check each of the fractions,which were then combined based on the similarity of their composition.The combined fractions were evaporated to remove solvent, dried andtheir tyrosinase inhibition activity measured as described in Example 2.It was found that fractions (P0346-HTP-F2-P0346-HTP-F4) were the mostactive and these fractions were combined and labeled as BK-F2-4. Aftersolvent evaporation, BK-F2-4 was further separated on a pre-packedreverse phase column (C-18 column) using a water/MeOH gradient. Eighteencompound peaks were observed following separation. Fourteen reversephase columns were performed and the similar fractions from each runwere combined. One compound peak referred to as UP288 in the combinedand enriched fraction showed strong tyrosinase inhibition activity (FIG.4 ). After separation and purification with preparative HPLC, 6 mg of1-(2-methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane(UP288) (1) was obtained. The structure of this compound was elucidatedusing MS and NMR spectroscopy (1H, ¹³C, HMQC and HMBC). FIG. 5 depictsthe chemical structure and ¹³C-NMR spectrum of UP288. UP288 is aninhibitor of tyrosinase having activity comparable with that of kojicacid with IC₅₀ value of 24 μM. FIG. 6 illustrates tyrosinase inhibitorydose response curves and IC₅₀ values for UP288 and kojic acid.

1-(2-Methoxy-4-hydroxyphenyl)-3-(2′-hydroxy-5′-methoxyphenyl)-propane(UP288). Yield 0.006% (purity >96%, HPLC); UV λ_(max): 281.0 nm; MS(Super Sound Ionization, Positive ion detection): m/z 289 (M+1, 100%);¹H-NMR (400 MHz, (CD₃)₂SO): δ 1.70 (2H, m, CH₂), 2.46 (4H, m, 2 CH₂),3.68 (3H, s, OCH₃), 3.73 (3H, s, OCH₃), 6.26 (1H, q, H-5), 6.35 (1H, d,H-3), 6.55 (1H, q, H-14), 6.65 (1H, d, H-13), 6.72 (1H, d, H-16), 6.86(1H, d, H-6), 8.69 (1H, s, OH), 9.20 (1H, s, OH); ¹³C-NMR (100 MHz,(CD₃)₂SO): Δ 28.5 (C-8), 31.6 (C-9), 34.5 (C-10), 55.0 (C-7), 55.6(C-17), 98.9 (C-3), 106.4 (C-5), 112.4 (C-16), 115.2 (C-13), 119.7(C-1), 119.8 (C-14), 120.3 (C-11), 120.4 (C-6), 132.9 (C-12), 144.6(C-4), 147.2 (C-17) & 158.3 (C-7).

Example 5 Extraction, Separation and Purification of1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2′,4′-Dihydroxyphenyl)-Propane(UP302a) (2) and1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2′,5′-Dihydroxyphenyl)-Propane(UP302b) (3) from Dianella ensifolia (P0389) (Whole Plant)

Dianella ensifolia (P0389, 300 g whole plant) was ground and extractedthree times 800 ml of MeOH:DCM (1:2). Dried extract (5 g) wasfractionated using a silica gel column with gradient solvent elution ofhexane/ethyl acetate (50/50) to MeOH. Fractions were collected in 2 setsof 264 test tubes. LC/MS/PDA was utilized to check each of thefractions, which were then combined into 22 fractions based on thesimilarity of their composition. (FIG. 7 ). The combined fractions wereevaporated to remove solvent, dried and their tyrosinase inhibitionactivity measured as described in Example 2. It was found that fractionsP0389-HTP-F12, P0389-HTP-F13 and P0389-HTP-F14 were the most active andthese fractions were combined and relabeled as DE-F12-14. After solventevaporation, DE-F12-14 was further separated on a pre-packed reversephase column (RP-column) using a water/MeOH gradient. Two major andeleven minor compound peaks were observed following separation. Thecompounds corresponding to each of these peaks were isolated following 7additional separations on RP-columns. All of the compounds collectedwere dried and tested for tyrosinase inhibitory activity. Two of theeleven minor peaks referred to as UP302a and UP302b, respectively,exhibited strong tyrosinase inhibitory activity. (FIG. 8 ). Afterseparation and purification, two active compounds were obtained:1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane(UP302a, 10 mg) (2) and1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,5′-dihydroxyphenyl)-propane(UP302b, 6 mg) (3). The structures of these two compounds wereelucidated using MS and NMR spectroscopy (1H, ¹³C, gHSQC and HMBC). FIG.9 depicts the gHSQC spectrum of UP302a. Tyrosinase inhibition assaysshowed that UP302a was the most potent inhibitor with an IC₅₀ of 0.24μM, while UP302b has an IC₅₀ of 12 μM.

1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,4′-dihydroxyphenyl)-propane(UP302a) (2). Yield 0.02% (purity >98%, HPLC); UV λ_(max): 279.8 nm; MS(Super Sound Ionization, Positive ion detection): m/z 303 (M+1, 100%);¹H-NMR (400 MHz, (CD₃)₂SO): δ 1.70 (2H, m, CH₂), 2.03 (3H, s, CH₃), 2.43(2H, m, CH₂), 2.49 (2H, m, CH₂), 3.58 (3H, s, OCH₃), 3.73 (3H, s, OCH₃),6.11 (1H, q, H-16), 6.25 (1H, d, H-14), 6.65 (¹H, d, H-5), 6.76 (1H, d,H-17), 6.97 (1H, d, H-6), 8.93 (1H, s, OH), 9.03 (1H, s, OH); ¹³C-NMR(100 MHz, (CD₃)₂SO): δ 28.8 (C-9), 29.3 (C-11), 31.1 (C-10), 55.3 (C-7),55.9 (C-8), 102.4 (C-14), 105.8 (C-16), 106.1 (C-5), 118.4 (C-1), 118.6(C-12), 126.9 (C-3), 127.0 (C-6), 130.1 (C-17), 155.7 (C-13), 156.2(C-15), 156.3 (C-4) and 156.8 (C-2).

1-(3-methyl-2,4-dimethoxyphenyl)-3-(2′,5′-dihydroxyphenyl)-propane(UP302b) (3). Yield 0.01% (purity >95%, HPLC); UV λ_(max): 279.8 nm; MS(Super Sound Ionization, Positive ion detection): m/z 303 (M+1, 100%);¹H-NMR (400 MHz, (CD₃COCD₃): δ 1.82 (2H, m, CH₂), 2.07 (3H, s, CH₃),2.52 (2H, m, CH₂), 2.56 (2H, m, CH₂), 3.63 (3H, s, OCH₃), 3.77 (3H, s,OCH₃), 6.64 (1H, q, H-15), 6.72 (1H, d, H-14), 6.64 (1H, d, H-5), 6.70(1H, d, H-17), 7.00 (1H, d, H-6), 7.65 (1H, s, OH), and 7.69 (1H, s,OH).

Example 6 Large-Scale Isolation of1-(3-Methyl-2,4-Dimethoxyphenyl)-3-(2′,4′-Dihydroxyphenyl)-Propane(UP302a) (2) from Dianella ensifolia (DE) (Whole Plant)

Dianella ensifolia (4.3 kg whole plant) was collected, ground andextracted three times using a percolation extractor with methanol as thesolvent. The extracts were combined and evaporated to remove themethanol. The crude extract was then suspended in water and partitionedwith DCM. The layers were separated and the DCM layer was evaporated toprovide 60 g of material. LC-MS/PDA analysis of both layers revealedthat the majority of the UP302a was present in the DCM layer with only aminor amount present in the water layer. The DCM extract wasfractionated on three separate silica gel columns eluting with agradient of hexane-ETOAC. A total of 15 sub-fractions were obtained andanalyzed by HPLC-MS/PDA. Targeted compound (UP302a) was found infractions 6 to 9, which were combined to yield a total of 3 g ofenriched UP302a. The enriched UP302a was further separated on an opencolumn packed with CG-161 resin eluting with a water-MeOH gradient. Atotal of 23 fractions were collected with the UP302a eluting infractions 15 to 21. Fractions 15-21 were then combined and the solventwas evaporated to yield 700 mg of a solid, which was further purified bypreparative HPLC on a C-18 column to generate 30 mg of UP302a. Thestructure, tyrosinase inhibitory activity and purity of the purifiedproduct was confirmed by NMR, enzyme inhibition assay and LC-MS/PDA.

Example 7 Synthesis of Diarylalkanes by Sodium Borohydride Reduction ofSubstituted Chalcones

A general method for the synthesis of diarylalkanes by sodiumborohydride reduction of substituted chalcones is described below usingthe reduction of 2,4-dihydroxy)-3′,4′-dimethoxychalcone (4) for purposesof illustration.

2,4-Dihydroxy-3′,4′-dimethoxychalcone (4) (40 mg) was dissolved in1-propanol (5 ml), followed by the addition of sodium borohydride (15mg) and the mixture was allowed to react at room temperature for 2hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) wasadded and the mixture was heated at 80° C. for 5 minutes and cooleddown. The mixture was then separated on a pre-packed C₁₈ column elutingwith a MeOH/H₂O gradient to provide1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-1-propanol (5). Thestructure of compound (5) was confirmed by MS, UV spectroscopy, 1D and2D ¹H-NMR.

1-(2,4-dihydroxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol (5). Yield60% (purity >98%, HPLC); UV λ_(max): 278.5 nm; MS (Super SoundIonization, Positive ion detection): m/z 305 (M+1, 100%); ¹H-NMR (400MHz, (CD₃)₂SO): δ 1.93 (2H, m, CH₂), 2.60 (2H, m, CH₂), 4.49 (1H, m,CH—OH), 3.78 (3H, s, OCH₃), 3.80 (3H, s, OCH₃), 6.28 (1H, q, H-5), 6.31(1H, d, H-3), 6.98 (1H, d, H-6), 6.71 (1H, q, H-5′), 6.77 (1H, d, H-2′),6.83 (1H, d, H-6′).

Using the above-described general method the following compounds werereduced to their corresponding alcohols:2,4-dihydroxy-2′-hydroxychalcone,2′-hydroxy-4′-methoxy-2,4-dimethoxy-chalcone,4′-hydroxy-4-hydroxy-chalcone, 2′,4′-dihydroxy-2-hydroxy-chalcone,2′,4′-dihydroxy-3,4-dimethoxy-chalcone,2′,4′,6′-trimethoxy-3,4-dimethoxy-chalcone and2′-hydroxy-4′-methoxy-3,4,5-trimethoxy-chalcone to provide1-(2,4-dihydroxyphenyl)-3-(2′-hydroxyphenyl)-1-propanol,1-(2-hydroxy-4-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol,1-(4-hydroxyphenyl)-3-(4′-hydroxyphenyl)-1-propanol,1-(2,4-dihydroxyphenyl)-3-(2′-hydroxyphenyl)-1-propanol,1-(2,4-dihydroxyphenyl)-3-(3′,4′-di-methoxyphenyl)-1-propanol,1-(2,4,6-trimethoxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol and1-(2-hydroxy-4-methoxyphenyl)-3-(3′,4′,5′-trimethoxyphenyl)-1-propanol.

Example 8 Synthesis of Substituted Diphenylpropanols by SodiumBorohydride Reduction of Substituted Diarylpropanones

A general method for the synthesis of substituted diphenylpropanols bysodium borohydride reduction of substituted diarylpropanones isdescribed below using the reduction of1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanone (6)for purposes of illustration.

1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanone (6)(5 mg) was dissolved in 1-propanol (1 ml), followed by the addition ofsodium borohydride (2 mg) and the mixture was allowed to react at roomtemperature for 5 hours. Upon completion of the reaction, 20% aceticacid (0.2 ml) was added to neutralize the excess sodium borohydride. Thereaction mixture was then separated on a pre-packed C₁₈ column elutingwith a MeOH/H₂O gradient to provide1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol (7).

Following the above-described general synthetic procedure the followingdiarylalkane compounds were reduced:1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanone,3-(5′-benzyloxy-4′-methoxy-2′-methylphenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)-1-propanone,1-(2-hydroxy-4-methoxyphenyl)-3-(2′,3′,4′,5′-tetrahydro-bezo(b)dioxocin-8′-yl)-1-propanoneand3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxyphenyl)-1-propenoneto provide1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol,3-(5′-benzyloxy-4′-methoxy-2′-methylphenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)-1-propanol,1-(2-hydroxy-4-methoxy-phenyl)-3-(2′,3′,4′,5′-tetrahydro-bezo(b)dioxocin-8-yl)-1-propenoland3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxy-phenyl)-1-propenol,respectively.

Example 9 Synthesis of 1,3-Bis(2,4-Dimethoxyphenyl)-Propan-1,3-Diol (9)

1,3-Bis(2,4-dimethoxyphenyl)-propan-1,3-dione (8) (5 mg) was dissolvedin 1-propanol (1 ml), followed by the addition of sodium borohydride (3mg) and the mixture was allowed to react at room temperature for 3hours. Upon completion of the reaction, 20% acetic acid (0.2 ml) wasadded to neutralize the excess sodium borohydride. The mixture was thenseparated on a pre-packed C₁₈ column eluting with a MeOH/H₂O gradient toprovide 1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol (9).

Example 10 Synthesis of1-(2,4,6-Trihydroxyphenyl)-3-(3′-Hydroxy-4′-Methoxyphenyl)-1-Propanol(11) from Neohesperidin

Neohesperidin is a glycoside of dihydrochalcone. A total weight of 100mg of neohesperidin was suspended in 10 ml of 1 N HCl and heated at 80°C. for 2 hours. The hydrolyzed product (10) was cooled down andextracted with ethyl acetate (3×10 ml). The ethyl acetate layers werecombined, evaporated to remove ethyl acetate and dissolved in 1-propanol(5 ml). Sodium borohydride (25 mg) was added to the propanol solutionand stirred at room temperature for 2 hours. After the completion of thereaction, the mixture was separated on a pre-packed C₁₈ column elutingwith a MeOH/H₂O gradient to provide1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol(11).

Example 11 Extraction, Purification and Structure Modification of Butrinto Synthesize142,4-Dihydroxyphenyl)-3-(3′,4′-Dihydroxyphenyl)-1-Propanol (14)

Butrin is a high content flavanone-glycoside that has been extractedwith methanol from the dried flowers of Butea frondosa and purified bymultiple reverse phase column chromatographic separations. Afterremoving sugars by hydrolysis with HCl, butin (12) was produced andpurified by RP-HTP (1.5% yield from the whole plant). Butin was thentreated with 10% sodium hydroxide at 80° C. to obtain butein (13), whichwas reduced with sodium borohydride to obtain1-(2,4-dihydroxyphenyl)-3-(3′,4′-dihydroxyphenyl)-1-propanol (14)(IC₅₀=250 nM).

Example 12 Synthesis of1-(2,4-Dihydroxyphenyl)-3-(3′-Methoxy-4′-Hydroxyphenyl)-1-Propanol (19)

Resorcinol (15), 3-methoxy-4-hydroxy-cinnamic acid (16) and H₂SO₄ (5%)were refluxed in THF for 4 hours to provide 7,4′-dihydroxy-3′-methoxyflavanone (17) (90% yield). The product, 7,4′-dihydroxy-3′-methoxyflavanone (17) was then treated with 10% sodium hydroxide at 80° C. for1 hour, followed by reduction with sodium borohydride in propanol toprovide, as confirmed LC-MS/PDA detection,1-(2,4-dihydroxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol (19).The crude product exhibits quite strong tyrosinase inhibitory activity.The mixture was further purified by HTP.

Example 13 IC₅₀ Measurements of Tyrosinase Inhibition by SyntheticDiarylalkanes

Inhibition of tyrosinase by synthetic diarylalkanes was measured usingthe method described in Example 2. The IC₅₀ value of each sample wascalculated using kinetics software to verify that the reaction waslinear at a specified time and concentration. Using the methodsdescribed in Examples 7-12a total of 24 compounds were synthesized andevaluated for their ability to inhibit tyrosinase. The results are setforth in Table 2.

TABLE 2 IC₅₀ values of synthetic diarylalkanes and/or diarylalkanolsTyrosinase Inhibition Compound Name (IC₅₀)1-(2,4-dihydroxyphenyl)-3-(3′,4′-dihydroxyphenyl)-1-propanol 0.5 μm1-(2,4-dihydroxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol 0.85 μm1-(2,4-dihydroxyphenyl)-3-(2′-hydroxyphenyl)-1-propanol 0.7 μm1-(2,4-dihydroxyphenyl)-3-(2′-methoxyphenyl)-1-propanol 3 μm1-(2,4-dihydroxyphenyl)-3-(4′-methoxyphenyl)-1-propanol 6 μm1-(2,4,6-trihydroxyphenyl)-3-(4′-aminophenyl)-1-propanol 8 μm1-(2,4-dihydroxyphenyl)-3-phenyl-1-propanol 8 μm1-(2,4-dihydroxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol 8.5μm1-(2-hydroxy-4-methoxyphenyl)-3-(3′,4′,5′-trimethoxyphenyl)-1-propanol11 μm 1-(2-hydroxy-4-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol25 μm 1-(2-hydroxy-5-methoxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol30 μm 1-(2,4-dihydroxyphenyl)-2-(4′-methoxyphenyl)-1-ethanol 77 μm1-(2-hydroxy-4-methoxyphenyl)-3-(2′,3′,4′,5′-tetrahydro-benzo(b)dioxocin-8′-yl)-72 μm 1-propanol3-(5′-chloro-1′-methyl-1′-hydro-imidazol-2′-yl)-1-(2-hydroxy-4-methoxyphenyl)-225 μm 1-propanol 1-(4-hydroxyphenyl)-3-(4′-hydroxyphenyl)-1-propanol305 μm1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3′-methoxy-4′-hydroxyphenyl)-1-propanol375 μm 1-(2,4-dihydroxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-ethanol 431μm 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane 700 μm1-(2-hydroxy-5-methoxyphenyl)-3-(2′,4′-dimethoxyphenyl)-1-propanol 1000μm 1-(2,4-dihydroxyphenyl)-2-(2′,4′-dichlorophenyl)-1-ethanol 1000 μm1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol1200 μm 1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol 1200 μm1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol1200 μm 1-(2,4,6-trimethoxyphenyl)-3-(3′,4′-dimethoxyphenyl)-1-propanol1500 μm

Example 14 Enzyme Inhibition Kinetics

Using the method described in Example 2, the inhibition of tryosinasewas evaluated at different concentrations (0, 261, 522, 1044 nM) of aninhibitor (UP302a) using L-DOPA at concentrations of 0.75, 1.25, and 2.5mM as the substrate. As shown in FIG. 10 , it was found that UP302a is acompetitive inhibitor with potent and long lasting inhibitory effect.Tyrosinase activity was not resumed for several days after incubationwith UP302a. In contrast, tyrosinase activity was totally restored afteronly 1 hour following incubation with kojic acid.

Example 15 Inhibition of Melanin Production from B-16 Cell Line

The inhibition of melanin production was evaluated using two differentassays. In the first assay, the inhibition of melanin production wasevaluated without induction by β-MSH; whereas in the second assay theinhibition of melanin production was measured with β-MSH induction incell culture medium. Briefly, B16 F1 cells (ATCC #CRL-622) were grown toconfluency and then seeded at 40,000 cells per well. The cells wereallowed to attach overnight at 37° C. in a humidified environmentcontaining 5% CO₂. On day 2, inhibitors were added to the cells atconcentrations ranging from 0-1000 μM in triplicate and allowed toincubate for four days. The amount of β-MSH required to induce melaninformation was determined by the addition of hormone at concentrationsranging from 0-1000 nM in ten-fold increments. Once the proper β-MSHconcentration was determined, cells were seeded as above, allowed toattach overnight and then co-incubated with tyrosinase inhibitors atconcentrations ranging from 0-1000 μM. Color development was visuallymonitored each morning. After the development of color, 200 μl of cellsupernatant was removed from each well and absorbance was measured at450 nm. The resulting readings were used to determine the IC₅₀ formelanin formation in the cell assay with and without β-MSH induction.For an initial comparison of cell toxicity, the 250 μM treated wellswere used to perform a lactate dehydrogenase assay (LDH). LDH is ametabolic enzyme that leaks out of damaged or dead cells. The enzymeconverts a chromophore in the presence of NAD⁺ to yield a color changethat can be monitored spectrophotometrically.

The results of this assay revealed that all of the natural inhibitorstested (UP288, UP302a, and UP302b) are at least as good, if not betterinhibitors than kojic acid. There were some differences in the IC₅₀values under the two sets of conditions. Inhibition by kojic acidimproved from an IC₅₀ of 170 μM for the endogenous experiment to an IC₅₀of 67 μM in the induced experiment. Of the inhibitors tested relative tokojic acid, compound UP302b was only one that that showed an increase inIC₅₀ under the two sets of conditions increasing from an IC₅₀ of 5.2 μMto an IC₅₀ of 34 μM. The IC₅₀'s measured for inhibition of tyrosinasewere relatively the same for all of the compounds tested with theexception of the two compounds UP302 and UP302b, which had low IC₅₀'s of0.2 μM and 0.3 μM, respectively, compared to 28 μM and 5.2 μM in theendogenous assay and 40 μM and 34 μM in the induced assay. Thesedifferences may be due to decreased cell penetration by UP302a (2) andUP302b (3), as compared to the other inhibitors. This is overcome,however by the strength of their inhibition of the enzyme.

Table 3 provides the results of these two assays for inhibitors UP288and UP302a relative to kojic acid.

Example 16 Cell Toxicity Assay

The compound treated wells were used to perform a lactate dehydrogenaseassay (LDH). LDH is a metabolic enzyme that leaks out of damaged or deadcells. The enzyme converts a chromophore in the presence of NAD′ toyield a color change that can be monitored spectrophotometrically. Thecytotoxicity was examined at a concentration of 250 μM. At thisconcentration none of these compounds are significantly more cytotoxicthan kojic acid. It should be noted however, that cytotoxicity at onlyone concentration (250 μM) was tested. As shown in the Table 3, UP288(1) and UP302a (2) showed moderate cytotoxicity, which were comparablewith kojic acid.

TABLE 3 Inhibition of mushroom tyrosinase and melanin formation in mouseB16 F1 cells by isolated compounds and comparison of cell toxicityEndogenous MSH Induced Tyrosinase Melanin Melanin Cell InhibitionInhibition Inhibition Toxicity Compound IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM)(LDH) UP288 24.0 108 105 0.315 UP302a 0.24 28 40 0.265 Kojic acid 29 17067 0.260

Example 17 Molecular Mechanics (MM2) Calculation

Molecular mechanics calculations were performed using Chem3D softwarefor purposes of energy minimization and determination of the most stable3-D conformation. The following parameters were used: Step interval=2.0fs, frame interval=10 fs, terminate after 10,000 steps, heating/coolingrate=1.000 Kcal/atom/PS, target temperature=3000K. Properties: pi bondorders and steric energy summary. All natural and synthetic compoundsand other diarylalkane and diarylalkanol structures were analyzed. Itwas found that the most potent tyrosinaseinhibitor—1-(3-methyl-2,4-dimethoxyphenyl)-3-(2,4-dihydroxyphenyl)-propane(UP302a (2), IC₅₀=0.24 μM)—isolated from whole plants of Dianellaensifolia (L.) DC. has a very unique 3-dimensional conformation in whichthe two aromatic rings were superimposed on each other. The minimizedtotal energy for the conformation is −4.7034 KJ/Mol. The distancebetween the two aromatic rings was 3.28 Å. The phenolic hydroxyl groupson the first aromatic ring were right above the two methoxyl groups onthe second aromatic ring with the distance between two oxygen atomsbeing 2.99 and 3.16 Å, respectively as illustrated in FIGS. 12-14 . Thisintramolecular parallel conformation allows this compound to perfectlychelate both copper ions of the binuclear enzyme when it is in theperoxide form [Cu^(II)—O₂—Cu^(II)] from both the top and the bottom.

Example 18 Formulation of the Diarylalkane Composition into a Cream

UP302a is comprised of a substituted diarylpropane as the major activecomponent. These compounds are soluble in high polarity solventsincluding, but not limited to ethanol, propylene glycol and ethyleneglycol. They can be formulated with a pharmaceutically and/orcosmetically acceptable excipient, an adjuvant, and/or a carrier.Examples of such excipients include, but are not limited to water,buffers, saline, Ringer's solution, dextrose solution, mannitol, Hank'ssolution, preservatives and other aqueous physiologically balanced saltsolutions. Nonaqueous vehicles including, but not limited to fixed oils,sesame oil, ethyl oleate, or triglycerides may also be used. Otheruseful formulations include, but are not limited to suspensionscontaining viscosity enhancing agents, including, but not limited tosodium carboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives or preservatives, such asantioxidants that enhance color and chemical stability. UP302 also canbe prepared in a liposome formulation to increase its skin penetrationor as a controlled release formulation, which slowly releases thecomposition of the active ingredient into the host.

UP302a is preferably administered topically as an ointment, gel, lotion,or cream base or as an emulsion, a patch, dressing or mask, anonsticking gauze, a bandage, a swab or a cloth wipe. Such topicalapplication can be locally administered to any affected area, using anystandard means known for topical administration. UP302 can beadministered to both humans and animals.

A therapeutic composition of UP302a can be administered in a variety ofunit dosage forms depending upon the method of administration andtargeted indications. An efficacious, nontoxic quantity is generallyrecommended in the range of 0.01% to 5% based on total weight of thetopical formulation. Two different concentrations of UP302a (0.01% and0.5% by weight) were formulated in creams as illustrated in Tables 4 and5. To prepare these creams the diarylalkane was dissolved in water atroom temperature and homogenized in a blender until it was fullydispersed in solution (approximately 5 minutes) to yield a compositionA. At room temperature and without stifling or agitating, Ultrez-21carbomer was added to the homogenized solution by sprinkling it onto thesurface and allowing it to fully wet (no white areas visible) and fallinto the solution. With gentle stifling, the solution was then heated to40° C. and glycerin was added and the composition was mixed for anadditional 5 minutes to provide Composition B. At 40° C., Composition Ais added to Composition B and the composition is mixed well untilhomogenous (approximately 5 minutes). The resulting emulsion is cooledto 30° C. and the pH adjusted to approximately 5.5 (5.3 to 5.7) bytitrating with neutralizer while stifling with a stir bar and/orspatula. The emulsion became highly viscous due to theneutralization-induced conformational change of the carbomer. Uponstifling the emulsion will achieved a suitable viscosity for an emulsioncream. The composition was mixed until uniform, poured into cleanstorage vessels and stored at 2° C. to 8° C.

TABLE 4 Composition of 0.01% Diarylalkane Cream Phase Ingredient % (w/w)Weight (g) Aqueous Water, Purified 85.00 12 Diarylalkane (UP302a) 0.010.0015 Ultrez 21 Carbomer 0.50 0.075 Glycerin 8.00 1.2 Oil PEG-7Glyceryl Cocoate 3.00 0.45 Caprylic/Capric Triglyceride 2.67 0.4 PHNeutralizer Sodium Hydroxide (18% w/v), 0.00 0.0 Molecular Biology GradeSUM 7 Ingredients 100 15

TABLE 5 Composition of 0.1% UP302 Cream Phase Ingredient % (w/w) Weight(g) Aqueous Water, Purified 84.00 12.6 Diarylalkane (UP302a) 0.1 0.0015Ultrez 21 Carbomer 0.50 0.075 Glycerin 8.00 1.2 Oil PEG-7 GlycerylCocoate 3.00 0.45 Caprylic/Capric Triglyceride 2.67 0.4 PH NeutralizerSodium Hydroxide (18% w/v), Molecular Biology Grade SUM 7 Ingredients99.7 15

The invention claimed is:
 1. A method for treating skin pigmentation dueto inflammatory conditions, the method comprising administering aneffective amount of a composition comprising a compound, wherein thecompound is of1-(2,4,6-trihydroxyphenyl)-3-(3′-hydroxy-4′-methoxyphenyl)-1-propanol,1,3-bis(2,4-dimethoxyphenyl)-propan-1,3-diol.
 2. The method of claim 1,wherein the administration lightens dark skin pigmentation, reduces skindullness, evens skin tone or corrects skin discoloration.
 3. The methodof claim 1, wherein the administration reduces signs of aging on theskin and reduces wrinkles associated with aging by neutralization ofactive oxygen species.
 4. The method of claim 1, wherein theadministration inhibits synthesis of melanin.